+

WO1999004811A1 - Systeme artificiel de production de virus infectieux du papillome humain (hpv) - Google Patents

Systeme artificiel de production de virus infectieux du papillome humain (hpv) Download PDF

Info

Publication number
WO1999004811A1
WO1999004811A1 PCT/US1998/015176 US9815176W WO9904811A1 WO 1999004811 A1 WO1999004811 A1 WO 1999004811A1 US 9815176 W US9815176 W US 9815176W WO 9904811 A1 WO9904811 A1 WO 9904811A1
Authority
WO
WIPO (PCT)
Prior art keywords
papiuomavirus
cells
dna
transfected
hpv18
Prior art date
Application number
PCT/US1998/015176
Other languages
English (en)
Other versions
WO1999004811A9 (fr
Inventor
Craig M. Meyers
Original Assignee
The Penn State Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Penn State Research Foundation filed Critical The Penn State Research Foundation
Priority to AU85802/98A priority Critical patent/AU8580298A/en
Publication of WO1999004811A1 publication Critical patent/WO1999004811A1/fr
Publication of WO1999004811A9 publication Critical patent/WO1999004811A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • Papillomaviruses are associated with greater than 90% of all cases of cervical cancers.
  • HPV human papiUomavirus
  • HPVs human papillomaviruses
  • HPVs human pathogenic papillomaviruses
  • the ability to manipulate the virus for use in diagnostic assays or vaccines has been hampered by the inability to propagate the virus in tissue culture. However, some progress has been made.
  • papiUomavirus replicates its genome to approximately 50 to 200 copies and maintains this copy number in basal ceils during the productive life cycle of the virus.
  • a basal cell divides, one of the daughter ceils migrates upwards and begins the process of terminal differentiation.
  • the signal transduction and gene expression pathways that are elicited during the differentiation process regulate viral gene expression.
  • papiUomavirus genes are expressed, viral DNA is amplified, viral late proteins are translated, and virion morphogenesis occurs.
  • Papillomavirions are sloughed off with the squames of the outermost epidermal layer and can then infect another host. Because of this dependency on terminal epithelial differentiation for replication, papillomaviruses cannot grow in ordinary monolayer culture; this limitation has hindered studies on the papiUomavirus life cycle.
  • the organotypic (collogen raft) tissue culture system accurately mimics an in vivo cellular environment and architecture (Ozbun and Meyers, 1996). Uses of the system are exemplified by investigations of the TGF- ⁇ 1 response of HPV-positive keratinocytes derived from neoplastic cervical biopsies.
  • TGF- ⁇ 1 transforming growth factor ⁇ 1
  • TGF- ⁇ 1 is a potent growth inhibitor for a variety of cultured cells.
  • organotypic raft cultures some of these conflicts were elucidated (Ozburn et al., 1996).
  • New methods are needed for in vitro viral propagation systems in particular for production of infectious viruses.
  • Infectious papiUomavirus is synthesized in an artificial, in vitro system.
  • the artificial system of infectious papiUomavirus includes a dermal equivalent, cells derived from differentiating epithelium and transfected with papiUomavirus DNA, a support for the transfected cells and conditions suitable for the transfected cells to produce papiUomavirus.
  • Preferred differentiating epithelial cells are keratinocytes.
  • infectious HPV18 is synthesized in differentiating epithelium transfected with the viral DNA.
  • a method for producing infectious papiUomavirus in the artificial system includes the following steps: obtaining a dermal equivalent, contacting the surface of the dermal equivalent with cells of a cell line derived from differentiating epithelium e.g. keratinocytes, and transfected with papiUomavirus HPV DNA to form a cellular composition, transferring the composition to a support; and providing a suitable environment allowing the papiUomavirus to be produced.
  • a dermal equivalent is generally constructed of a mixture of type I collagen and fibroblasts.
  • the transfecting DNA includes a mutant DNA. Same mutant DNAs lead to production of attenuated viruses useful for making vaccines.
  • a method for investigating the effects of a composition, e.g. a candidate inhibitor, on HPV positive cells comprises obtaining HPV positive cells in the artificial system of the present invention, contacting the cells with the composition, and observing the subsequent growth and development of the contacted cells to determine if there is an effect of the composition based on comparisons with and without the composition.
  • Efficient in vitro synthesis of infectious HPV is optimized with a cell population homogenous in copy number and maintenance of episomai viral DNA.
  • Human papillomaviruses can be propagated in vitro in quantities sufficient to investigate questions concerning the biology of the infectious process of the virus. This tissue culture system can be used to propagate any papiUomavirus type if the viral DNA is available.
  • viral particles produced in this system can be used to test a patient's serum to determine whether there is evidence of an infection and, if so, to determine the viral types(s) involved in the infection.
  • Mutant papillomaviruses can also be produced using the system and methods of the present invention. Because DNA is the starting point, mutations can be introduced specifically and easily into known genes or functional regions. This mutated viral DNA can be made in large quantities and used for transfection to make more virus. Chimeras, i.e. mixture of DNA from different sources, can also be made. Mutant viruses are useful to investigate the mechanisms of the viral life cycle, virion morphogenesis, and infectivity.
  • Attenuated viruses are viruses that have been weakened by genetic manipulation, in their ability to cause disease (an example is the polio vaccine). The virus is still capable of infection but is limited in its pathogenicity. During infection the immune system mounts a response. The attenuated virus is cleared either due to it being defective and incapable of maintaining the infection, or cleared by the immune system. Boosters may be needed to increase the immunity. Defective viruses cannot grow. Aspects of the invention include the development of anti-viral therapies.
  • FIG. 1 depicts examination of HPV18 DNA transfected HFK raft cultures for the presence of virus particles;
  • FIG. 1A shows a representative nucleus with numerous virions scattered throughout intranuclear space (arrows point to representative viral particles; bars equal 100 nm).
  • FIG. 1B is an enlargement of a second representative nucleus containing virions (arrows point to representative viral particles; bars equal 100 nm).
  • FIG. 2 illustrates Southern (DNA) blot hybridization of HPV18 DNA- transfected human ectocervical keratinocytes (HCK) subcloned cell lines.
  • FIG. 3 shows HPV18 virions banded on isopycnic gradients. HPV18
  • FIG. 4 illustrates Southern blot hybridization of fractions collected from virus preparations of HPV18 DNA-transfected subcloned line HCK 18: IBs raft cultures.
  • FIG. 5 shows electron micrographs of HPV18 virions approximately 50 nm in diameter.
  • the concentration of virions produced in the subcloned line HCK 18:1 Bj resulted in the clumping of the virions as seen in FIG. 5A and B, (arrows point to representative viral particles). Bars in (A) and (B) equal 50 nm.
  • Purified virions from subcloned cell lines were diluted 20-fold in TE buffer [pH 8.0] to prevent clumping; individual particles were observed in FIG. 5C,E,F.
  • FIG. 5C shows results in subclone line HCK 18:1 Bj
  • FIG. 5D shows results in parental line HCK 18:1B (undiluted preparation).
  • FIG. 5E-F shows results in subclone line HCK 18: I Bs.
  • the magnification in FIG. 5F is approximately 2x that of FIG. 5C-E. All virus particles are approximately 50 nm in diameter.
  • FIG. 6 illustrates identification of HPV18 specific spliced transcripts (RNA) in HPV18 infected keratinocytes. Placement and orientation are illustrated by open arrows.
  • HPV18-specific spliced transcripts identified in the HCK 18 : 1 B j cell line and in the infected keratinocytes are labeled -ft, *, >, +, and ⁇ .
  • HPV human papiUomavirus
  • the invention relates production of infectious virus of a preselected HPV type by transfecting differentiating epithelial cells, e.g., human ectocervical keratinocytes (HCK) and human foreskin keratinocytes (HFK), with HPV DNA.
  • epithelial cells e.g., human ectocervical keratinocytes (HCK) and human foreskin keratinocytes (HFK)
  • HCK human ectocervical keratinocytes
  • HFK human foreskin keratinocytes
  • the maintenance of episomal HPV18 viral genomes in a system of the present invention was confirmed by Southern (DNA) blot hybridization.
  • Cell lines stably maintaining episomal copies of HPV18 DNA were subsequently allowed to grow as stratified and differentiated epithelial tissues in an organotypic culture system. Electron microscopy of thin-sections of organotypic culture tissue demonstrated nucle
  • FIG. 1A shows a representative nucleus with numerous virions scattered throughout intranuclear space (arrows point to representative viral particles).
  • FIG. 1B is an enlargement of a second representative nucleus containing virions (arrows point to representative viral particles). Bars equal 100 nm. The presence of virus particles was limited to the upper suprabasal layers of the epithelium, and the particles observed were similar to those detected in clinical biopsy material from low-grade lesions (Shally et al., 1996). Results obtained were reproducible using several HPV18 DNA-transfected HCK and HFK lines. 1.
  • HPV18 DNA linear copies of HPV18 DNA were electroporated with a selected marker into foreskin and ectocervical primary keratinocyte cell lines.
  • a Hygromycin B selectable marker was used, but any selectable marker is suitable i.e. G418.
  • the DNA was prepared by standard DNA preparation protocols, then it was EtOH precipitated and resuspended and used at a concentration of 1 ⁇ g/10 ⁇ l.
  • Foreskin and ectocervical keratinocytes were isolated from newborn circumcision and adult hysterectomy tissue specimens, respectively.
  • the dermis was removed from the epidermis and discarded.
  • the epidermis was cut into approximately 2-mm square pieces (tissue "chips"), placed into a 10- cm tissue culture dish and allowed to partially dry at room temperature. Culture media was added to amply cover the tissue, and samples were incubated at 37° C. Media were changed every other day. Explants were observed daily for outgrowth of cells from the tissue chips. After outgrowth of cells began, mitomycin C-treated J2 3T3 (Rheinwald and Green, 1975) cells were prepared and added to the explant cultures.
  • Adding mitomycin C treated cells at this point differs from previously published techniques. This procedure maintains cells in an environment more similar to the in vivo environment, allowing them to maintain their in vivo phenotype. When outgrowths covered a third of the plate they were trypsinized and frozen for long term storage.
  • Cells that obtained DNA via the electroporation procedure were selected by adding between 10 and 25 ⁇ g of Hygromycin B per ml of media beginning about 48 to 72 hours after electroporation. Selection was continued until killing of the cells was observed as determined when cell formed numerous vacuoles round up and float off the plate.
  • the amount of time of treatment with Hygromycin B is empirical based on observations that are routine. Monolayers were passaged and frozen at different passages.
  • Passage number does not appear to be a crucial factor as long as the viral DNA stays episomal as determined by Southern blot. However, cells passaged over twenty times are not preferred because they may begin to lose their ability to differentiate.
  • Cells were frozen at a concentration of 1-2 x 10 6 per ml in epithelial freezing media. Freezing media contains standard E media with twice the amount of fetal calf serum, 15% fetal calf serum, and 22% glycerol. This freezing media is known to those of skill in the art for freezing primary keratinocytes. Standard E media was used but with and without EGF (see protocol herein). Serum lots were tested for best differentiation results. 2.
  • the cells were analyzed at different passages by Southern blot analysis. Cells were preferably analyzed at passages 3 and 4. Many cell lines were observed that had viral DNA episomally maintained. Episomal viral DNA was observed in cell lines from passage 1 to passage 12.
  • the parental (transfected lines) were subcloned, then analyzed by Southern blot. Virus were purified from the parental lines electroporated with HPV18 DNA, but with minimal success. Southern blots representing some of the sub graduates were made. Cell lines were heterogeneous as to their maintenance and copy number of HPV DNA. The parental lines were subcloned, which improved the results. Subcloning is a key factor in success of the artificial system, because the productivity varied among sub graduates and was generally greater than in the parental line.
  • Subcloned lines used for further analysis contained 100-200 copies of episomal HPV18 DNA. Two of the subations used were designated "s" and "j".
  • Raft cultures were grown for 12 days at an air-liquid interface of the cultures using standard E-media.
  • Changes made in the organotypic culture system used for the present invention include: (i) 2.4 ⁇ l of 10N NaOH per ml of coilagen mix is added; (ii) the pH of the collagen matrix mix is increased by using collagen at a specific concentration (4mg/ml); and using a colorimetric change from yellowish-red to light red as an indicator; (iii) the wire grids are bent so that they will allow using between 10 and 12 ml of media; this makes the grids sit at a specific height above the plastic tissue culture plate and supplies ample nutrients that may be lacking when less collagen mix is used.
  • HCK 18:1 B A parental line that was subcloned is designated HCK 18:1 B (HCK refers to "human ectocervical keratinocytes").
  • Southern blot hybridization analysis demonstrated that subcloned cell lines differed in their episomal HPV18 DNA copy number (FIG. 2). This result suggested that parental cell lines consisted of a heterogeneous population of cells with respect to HPV18 DNA content. Subcloned cell lines were identified alphabetically following the parental cell line designation. Results from representative subcloned lines HCK 18:1 Bt, s, k, I, and j were chosen to demonstrate the heterogeneity of the parental cell lines in their copy number and maintenance of episomal viral DNA.
  • Lanes designated 1000, 100, and 10 represent copy number controls of viral genomic DNA per 10 6 cells. Lanes t, s, k, I, and j, 5 ⁇ g total DNA isolated from subcloned cell lines was electrophoresed per lane in a 0.7% agarose gel. Southern blot hybridization was performed. The lowest band indicated Form I (supercoiled) viral DNA. The two upper bands indicated multimeric forms of viral DNA common to papiUomavirus. Arrows on the left indicate the position of molecular size markers, that were ⁇ DNA Hind ⁇ digested in bp; 23,130; .9,416; 6,557; 4,361; 2,322 and 2,022.
  • Cesium chloride gradients of virions were prepared from the raft cultured tissues. Gradients were used to purify the virions. Using the subcloned lines, the protocols for purification included conditions for fractionating the gradient, removing specific volumes from one end of the gradient. Hoefer Scientific gradient tube fractionator (FS101), capable of fractioning 0.5ml samples was used. Southern blot analysis of fractions from the gradient were analyzed by the standard Southern blot technique. 3 to 5 x 10 9 particles were purified. Fractions shown to be positive for viral DNA were dialyzed against multiple changes of TE buffer (pH 7.6). Virus was purified from the parental and subcloned cell lines.
  • Tissue was ground in a mortar with sea sand, resuspended in buffer #1 (1 M NaCI, 0.05 MNa 2 HPO 4 [pH 7.4]), and centrifuged for 10 min at 8,000g. The supernatant was removed and saved on ice; the pellet was reextracted with buffer #1 , and centrifuged for 10 min at 8,000g. Supernatants were pooled, CsCI was added to a final concentration of 1.3 g/ml, and centrifuged for 24 hours at 135,000g. Arrows indicate bands of HPV18 virions (between 1.3 to 1.4 g/cm 3 ) observed in two typical examples.
  • FIG. 3 Fractions of 0.5 ml each were collected from the gradient and dialyzed against TE buffer [pH 8.0]. DNA was purified from each fraction, and Southern blot hybridization was performed. Fractions 7, 8, and 9 of FIG. 4 were found to contain HPV18 DNA at high levels. Fractions positive for HPV18 DNA correlated to fractions where the banded virus was visually observed in the gradient (FIGS. 3,4). HPV18-DNA positive fractions contained virus particles as detected by electron microscopy (FIG. 5). Gradient fractions determined to be positive for HPV18 DNA by Southern blot hybridization were stained with phosphotungstic acid and examined by electron microscopy.
  • the concentration of virions produced in the subclone line HCK 18 : 1 B j resulted in the clumping of the virions as seen in FIG. 5A and B, (arrows point to representative viral particles). Bars in (A) and (B) equal 50 nm.
  • Purified virions from subcloned cell lines were diluted 20-fold in TE buffer [pH 8.0] to prevent clumping; individual particles were observed (C,E,F).
  • C from subclone line HCK 18:1Bj
  • D from parental line HCK 18:1B (undiluted preparation);
  • E-F from subclone line HCK 18:1 Bs.
  • the magnification in (F) is approximately 2x that of (C-E).
  • HPV18 virions showed large amounts of HPV18. Purified virus was infectious as determined by using primary keratinocyte lines. (The parental line-derived virus were infectious, but fewer virions were produced.)
  • Standard primary human foreskin keratinocyte lines were prepared and maintained as described herein. Confluent 10 cm plates of cells were used. Samples of the virus were placed on the cells. Media was removed from the plates. Then, 50, 100, or 200 ⁇ l of viral stocks were added to 0.5 ml of culture media and this composition was added to the plates. Plates were incubated with the viral solutions for about 2 hours and were rocked (tilted plates back and forth by hand) every 15 minutes. Then 10 ml of culture media was added to each plate and plates were incubated for varying times before harvesting the RNA. The purified HPV18 virions were capable of infection, the final step in the viral life cycle in vitro.
  • Duplicate plates were also maintained by passaging infected cell lines. Data from Southern blot hybridization analysis indicate that viral inocula contained between 1-2 x 10 9 particles per ml for the subclonal cell lines HCK 18 : 1 B j and HCK 18: 1 Bs, and greater than one order of magnitude fewer particles for the parental line HCK 18:1 B.
  • HPV18 cDNAs were PCR-amplified using primers to an E6 nucleotide sequence (5'-TGAGGATCCAACACGGCGACC-3') and to an E4/E2 nucleotide sequence (5'-GGTGTAGCTGCACCGAGAAGTGG-3'); placement and orientation are illustrated by open arrows.
  • the cDNA products were electrophoresed in an 1.2% agarose gel followed by Southern blot hybridization.
  • RT-PCR-amplified cDNAs from the HCK 18 : 1 B j subclone were cloned. Sequencing revealed cDNAs corresponding to the HPV18 transcripts E6 * I,E7,E1 ⁇ E4 and E6 * lll.
  • HPV18-infected cell lines The life-span of HPV18-infected cell lines was assessed using separate viral preparations from the subcloned lines HCK 18:1Bj, HCK
  • HPV18 DNA was linearized at nucleotide 2440 by restriction digest with EcoRI interrupting the E1 open reading frame. Replication and episomal maintenance was dependent on precise recircuiarization of the viral DNA. Viral DNA was electroporated into primary human foreskin keratinocytes (HFK) or primary human ectocervical keratinocytes (HCK).
  • HFK primary human foreskin keratinocytes
  • HCK primary human ectocervical keratinocytes
  • Organotypic Raft Cultures An in vitro organotypic (raft) culture system capable of reproducing the differentiation-dependent replication cycle of human papiUomavirus is an aspect of the invention.
  • a dermal equivalent is made from a mixture of type I collagen and fibroblasts.
  • Cells of a line derived from differentiating epithelial cells such as a cervical intraepitheiiai neoplasia type 1 (CIN-1) are placed on top of the dermal equivalent and, while submerged, are allowed to grow to confluence.
  • the dermal equivalent with epithelial cells on top is then lifted onto a wire grid where it remains at an air-liquid interface.
  • Protocol for Purifying Virion Particles by Gradients Cells are washed with 0.15 M NaCI and ground in mortar with regular sand; the results of grinding are resuspended in about 25 ml of 1 M NaCI; 0.05M Na Phosphate Buffer at pH 8; the suspension is centrifuged using a ss34 rotor at 10-10.5 k rpm (8k x g) for 10 min at 4°C; the supernatant is saved; the pellet is reextracted in 25 ml 1M NaCI; 0.05 MNa Phosphate Buffer at pH 8; the suspension is centrifuged using a ss34 rotor at 10-10.5k rpm for 10 min at 4°C; the super ⁇ atants are pooled; to pellet virus, pooled supernatants are centrifuged (130 k x g) 43k x rpm using a type 60Ti rotor for 60 min at 4°C; or a SW41
  • CsCI is added to 1.3 g/ml to the pooled supernatants; and centrifuged at 135 k x g 39 k x rpm, using a VTi 65 rotor, 24 hr at
  • Formamide (50%) 15.0 ml 15.0 ml ⁇ l DNA (25-100 n ⁇ )
  • probe by calculating the amount of probe needed (10 6 cpm/ml of hybridization mix): 5 ⁇ l probe DNA; denature 10 min at 100°C, place immediately on ice, add all other compounds and incubate 30 min at 37°C. Stop reaction by adding 2 ⁇ l of 500 mM EDTA. Adjust volume to 100 ⁇ l and spin for 5 min in a Quick Spin Column. Count 2 ⁇ l. Add 50 ⁇ l of 10mg/ml denatured at 100°C ssDNA.
  • cells ml of E + 10% fcs with 5mM BES at pH 7.2 (from a 500 mM BES stock) 20 x 10 6 cells/mi 6. Mix 250 ⁇ cells (5 x 10 6 cells) with the DNA mix.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des systèmes artificiels de production de virus infectieux du papillome humain qui permettent le développement de stratégies thérapeutiques et diagnostiques basées sur des études relatives à l'infectivité et à la replication de maladies provoquées par ces virus. L'introduction artificielle d'un ADN génomique de virus du papillome dans une lignée cellulaire épithéliale, en particulier un ADN 18 génomique de type HPV, est transfectée en kératinocytes primaires. La transfection est suivie d'une expansion clonale des cellules transfectées, d'une induction de stratification éphitéliale et d'une différenciation dans des cultures organotypiques ayant pour résultat la synthèse des virions. Les particules de virus synthétisées, observées au microscope, présentent un diamètre d'environ 50 nanomètres. Les virions HPV 18 produits par le système et purifiés par un gradient isopycnique peuvent infecter des kératinocytes.
PCT/US1998/015176 1997-07-22 1998-07-22 Systeme artificiel de production de virus infectieux du papillome humain (hpv) WO1999004811A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU85802/98A AU8580298A (en) 1997-07-22 1998-07-22 Artificial system for the production of infectious human papillomavirus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/898,636 1997-07-22
US08/898,636 US5994115A (en) 1997-07-22 1997-07-22 Artificial system for the production of infectious human papillomavirus

Publications (2)

Publication Number Publication Date
WO1999004811A1 true WO1999004811A1 (fr) 1999-02-04
WO1999004811A9 WO1999004811A9 (fr) 1999-04-22

Family

ID=25409787

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/015176 WO1999004811A1 (fr) 1997-07-22 1998-07-22 Systeme artificiel de production de virus infectieux du papillome humain (hpv)

Country Status (3)

Country Link
US (1) US5994115A (fr)
AU (1) AU8580298A (fr)
WO (1) WO1999004811A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6841157B2 (en) * 2000-07-07 2005-01-11 Merck & Co., Inc. Production of chimeric human papillomavirus
US20110300530A1 (en) * 2010-06-02 2011-12-08 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. In vitro test system for viral infections

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6495361B1 (en) * 2000-07-21 2002-12-17 University Of Arkansas Method of producing infectious papillomavirus in placental cells
US6974697B2 (en) * 2001-03-02 2005-12-13 Stratech Corporation Skin substitutes with improved barrier function
ES2457092T3 (es) * 2001-03-02 2014-04-24 Stratatech Corporation Sustitutos de piel mejorados y usos de los mismos
WO2004081178A2 (fr) * 2003-03-06 2004-09-23 Indiana University Research And Technology Corporation Methodes et compositions pour produire des souches hpv infectieuses
EP1660632B1 (fr) 2003-08-01 2014-02-19 Stratatech Corporation Equivalents de peau humaine exprimant des polypeptides exogenes
US7285386B2 (en) * 2003-10-29 2007-10-23 University Of New Mexico, And Educational Institution Of The State Of New Mexico RhPV as a model for HPV-induced cancers
WO2006094070A2 (fr) 2005-03-01 2006-09-08 Stratatech Corporation Substituts de peau humaine exprimant des polypeptides exogenes
JP2008532550A (ja) 2005-03-17 2008-08-21 ストラタテック コーポレーション 純度が改良された代用皮膚
CN109666643B (zh) * 2018-12-24 2022-06-03 武汉大学 一株含有游离型hpv18的宫颈上皮内瘤样病变细胞系及其用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FRATTINI M. G., ET AL.: "IN VITRO SYNTHESIS OF ONCOGENIC HUMAN PAPILLOMAVIRUSES REQUIRES EPISOMAL GENOMES FOR DIFFERENTIATION-DEPENDENT LATE EXPRESSION.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93., 1 April 1996 (1996-04-01), US, pages 3062 - 3067., XP002913731, ISSN: 0027-8424, DOI: 10.1073/pnas.93.7.3062 *
MEYERS C., ET AL.: "BIOSYNTHESIS OF HUMAN PAPILLOMAVIRUS FROM A CONTINUOUS CELL LINE UPON EPITHELIAL DIFFERENTIATION.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 257., 14 August 1992 (1992-08-14), US, pages 971 - 973., XP002913730, ISSN: 0036-8075, DOI: 10.1126/science.1323879 *
MEYERS C., ET AL.: "TISSUE CULTURE TECHNIQUES FOR THE STUDY OF HUMAN PAPILLOMA VIRUSES IN STRATIFIED EPITHELIA.", CELL BIOLOGY: A LABORATORY HANDBOOK, XX, XX, vol. 01., 1 January 1998 (1998-01-01), XX, pages 513 - 520., XP002913734 *
MEYERS C.: "ORGANOTYPIC (RAFT) EPITHELIAL TISSUE CULTURE SYSTEM FOR THE DIFFERNTIATION-DEPENDENT REPLICATION OF PAPILLOMAVIRUS.", METHODS IN CELL SCIENCE., KLUWER ACADEMIC PUBLISHERS, AMSTERDAM., NL, vol. 18., 1 September 1996 (1996-09-01), NL, pages 201 - 210., XP002913733, DOI: 10.1007/BF00132885 *
OZBUN M. A., MEYERS C.: "TRANSFORMING GROWTH FACTOR BETA1 INDUCES DIFFERENTIATION IN HUMAN PAPILLOMAVIRUS-POSITIVE KERATINOCYTES.", JOURNAL OF VIROLOGY., THE AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 70., no. 08., 1 August 1996 (1996-08-01), US, pages 5437 - 5446., XP002913732, ISSN: 0022-538X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6841157B2 (en) * 2000-07-07 2005-01-11 Merck & Co., Inc. Production of chimeric human papillomavirus
US20110300530A1 (en) * 2010-06-02 2011-12-08 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. In vitro test system for viral infections
US8876903B2 (en) * 2010-06-02 2014-11-04 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. In vitro test system for viral infections

Also Published As

Publication number Publication date
WO1999004811A9 (fr) 1999-04-22
US5994115A (en) 1999-11-30
AU8580298A (en) 1999-02-16

Similar Documents

Publication Publication Date Title
Flores et al. Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line
Meyers et al. Synthesis of infectious human papillomavirus type 18 in differentiating epithelium transfected with viral DNA
Meyers et al. In vitro systems for the study and propagation of human papillomaviruses
Ruesch et al. Activation of papillomavirus late gene transcription and genome amplification upon differentiation in semisolid medium is coincident with expression of involucrin and transglutaminase but not keratin-10
Schmitt et al. The primary target cells of the high-risk cottontail rabbit papillomavirus colocalize with hair follicle stem cells
Frattini et al. In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.
Bollag et al. Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation
Bedell et al. Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation
Kawana et al. Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm
McLaughlin-Drubin et al. Propagation, infection, and neutralization of authentic HPV16 virus
Li et al. Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses
McLaughlin-Drubin et al. Human papillomavirus type 45 propagation, infection, and neutralization
US5994115A (en) Artificial system for the production of infectious human papillomavirus
Gius et al. Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester
Meyers et al. Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera
Fang et al. Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures
Christensen et al. Coinfection of human foreskin fragments with multiple human papillomavirus types (HPV-11,-40, and-LVX82/MM7) produces regionally separate HPV infections within the same athymic mouse xenograft
CN112048477A (zh) Ebv病毒感染人工呼吸道上皮模型的建立方法
Scherneck et al. The hamster polyomavirus—a brief review of recent knowledge
EP1309342B1 (fr) Production d'un papillomavirus humain chimerique
May et al. Study of the functional activities concomitantly retained by the 115,000 M r super T antigen, an evolutionary variant of simian virus 40 large T antigen expressed in transformed rat cells
Hurwitz et al. Immortalization of rat embryo fibroblasts by an adenovirus 2 mutant expressing a single functional E1a protein
Lehr et al. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation
JP2002535965A (ja) ヒトパピローマウイルス・ウイルス様粒子を使用した中和アッセイ
US20090148950A1 (en) Production of packaged dna sequences

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

AK Designated states

Kind code of ref document: C2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/6-6/6, DRAWINGS, REPLACED BY NEW PAGES 1/7-7/7; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载